1. Field of the Invention
The present invention relates to detecting the presence of endotoxins of Gram-negative bacteria in solid, liquid and gaseous samples. More specifically, the present invention is a method and apparatus for automatically and accurately infering the presence and type of Gram-negative bacterial endotoxins from solid, liquid and gaseous samples by measuring hydroxy fatty acid methyl esters of Lipid A lipopolysaccharides.
2. Background of the Invention
Pathogenic microorganisms are bacteria responding negatively to the Gram stain, hence the designation Gram-negative bacteria. The presence of Gram-negative bacteria stimulates a host of responses by the immune system of human beings. In particular, these bacteria are pyrogenic or fever producing. Endotoxic bacteria can also induce other responses including abortions, pulmonary edema, hemorrhagic pneumonia, uremia, anaphylactic shock, and death of the host body. Endotoxins can enter the body through the air or by injection and even slight traces of endotoxins provoke a response.
Part of the problem of avoiding exposure of humans to pathogenic bacteria is solved by being able to detect the presence of pathogenic organisms among nonpathogenic organisms. Fortunately, not only does their virulence distinguish them but also their biomolecular makeup. Scientists have looked to cellular lipids, in particular, for distinguishing microorganisms and have found that these lipids are distinctive not only of the genus but the species. Since lipids of Gram-negative bacteria are characteristic, identification of the lipid is tantamount to identifying the bacteria from which it came. This critical piece of information could greatly accelerate tracing the cause of an infection and thus hasten the treatment of the disease.
Within the cellular lipids of cells are cholesterol, triglycerides, phospholipids, quinones and lipopolysaccharides. The last of these, lipopolysaccharides or LPS, which are exclusively major components of Gram-negative bacterial cell walls, contain covalently bound hydroxy fatty acid lipids. The analysis of LPS hydroxy fatty acids, provides the sensitivity and selectivity needed for accurate identification. LPS, which contains the endotoxic constituents of Gram-negative bacteria, comprise a covalently linked lipid A component and a polysaccharide component, the latter being a chain of covalently linked monomeric units which form a large carbohydrate. The lipid A component of the LPS (which is thought to be responsible for the observed physiological reactions to endotoxins) will be used as a distinguishing biomolecule. Other cells, including fungi, protozoa, and other bacteria can be distinguished by the approximately 200 different types of extractible (without prior hydrolysis) phospholipid fatty acids, the most commonly used biomarkers.
The detection of covalently bound hydroxy fatty acid in the LPS provides definitive identification of Gram negative LPS lipid A endotoxins. This specificity allows classification to the family and in some cases species level amongst Gram-negative bacteria.
It is important that certain environments be as Gram-negative-bacteria-free as possible. For example, Gram-negative bacteria are the most common contaminants of water. Also, intravenous fluids, parenteral fluids, prostheses, artificial organs, organs for transplant, medical and veterinary procedure devices, eye and ear care products, foods, potable water, and water used in fabrication of semiconductors must be exceptionally free of bacteria before use. These environments must also be free of endotoxins, which can exist in the absence of viable bacterial cells.
Methods for identifying the presence and type of bacteria in general are not new. Biochemical techniques have been applied to assess microbial biomass in different environments. Some of these techniques require culturing of the organisms or quantitative removal of organisms from the sample and some do not. Procaryotic biomass, for example, can be accurately estimated from the quantity of muramic acid if the proportions of cyanophytes, Gram-negative and Gram-positive bacteria are known. These proportions can be determined by examining the cell wall with an electron microscope or by biochemical markers.
There are several methods used for detecting Gram-negative bacteria. In one, a sample is subjected to chloroform-methanol extraction (the so-called Bligh-Dyer extraction method). The lipid soluble fraction is subjected to mild alkaline methanolysis, then fractionated into polar lipid, glycolipid, and neutral lipid fractions via silicic acid chromatography. The polar lipid fraction, which contains the unique signature biomarkers, phospholipids, is then analyzed via capillary column gas chromatography. While bacterial identification to the species level is possible with this technique, endotoxins are not specifically detected.
Another test for the presence of Gram-negative bacteria is the rabbit pyrogen test, described more fully in U.S. Pat. No. 4,093,381, in which a rabbit is exposed to a potentially endotoxic sample and its body temperature monitored. This test is relatively insensitive, detecting about 5.times.10.sup.-8 grams LPS or 500,000 bacteria per milliliter, and is nonspecific; that is, it cannot distinguish between bacterial and nonbacterial pyrogens. Furthermore, this test is slow and expensive and the increasing concern for animal welfare raises ethical issues.
A third test for the presence of gram negative bacteria is the Limulus Amebocyte Lysate (LAL) test which uses an extract of the horseshoe crab. This method, described in U.S. Pat. No. 4,279,774, is relatively more sensitive than the rabbit pyrogen test, detecting about 10.sup.-11 grams LPS or about 1000 to 10,000 bacteria per milliliter. This test is subject to interferences when applied to fluids that contain proteins such as insulin, growth hormone, blood and urine, and is difficult to execute. A LAL test can be performed by three methods. They are the so-called clot assay, the chromogenic assay, and the kinetic assay. It, like the rabbit pyrogen test, is nonspecific in detecting endotoxin-producing bacteria, is costly and again raises the ethical issues associated with animal testing.
Other methods for detecting endotoxins are described in U.S. Pat. Nos. 4,758,509, 4,276,050, 4,195,225, 3,891,508, and 3,365,277.
There is a need for a fast method and apparatus for determining the presence and type of endotoxic LPS of Gram-negative bacteria with a high degree of accuracy and speed.